Project Summary Excitement about RNA triple helices has grown rapidly in recent years due to their newfound biological roles in telomere synthesis, pre-mRNA splicing, protecting cancer-promoting RNAs from degradation and now genome editing. Therefore, the discovery and characterization of more RNA triple helices is a pre-requisite to elucidating other fundamental biological processes mediated by triple-helical structures. As only 11 naturally occurring RNA triple helices have been validated to date and more are predicted, there is a clear need for the development of molecular tools to determine the global landscape of RNA triple helices throughout nature. Herein, the principal investigator proposes to characterize the MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) RNA triple helix and its protein-binding partner, METTL16 (methyltransferase-like protein 16). The MALAT1 triple helix and METTL16 complex is the first putative triple-stranded ribonucleoprotein complex, thereby representing a unique opportunity to provide unprecedented insights into triple-stranded RNA biology. In Research Areas #1 and #2, the structural basis of how METTL16 and small molecules specifically recognize and interact with the MALAT1 triple helix will be determined for the first time using a combination of X-ray crystallography and biochemical methods. This effort will generate the foundational knowledge needed to develop novel experimental tools for the global discovery of more RNA triple helices. Moreover, the discovery of an entirely new class of proteins, that is triple-stranded RNA-binding proteins, is anticipated. In Research Areas #3 and #4, the biological function of human METTL16 as a putative triple-stranded RNA-binding protein will be investigated using both in vitro and cell-based biochemical assays. This functional investigation will focus on the roles of the METTL16-MALAT1 triple helix complex as well as broader roles for METTL16 and its RNA-binding partners in the context of Miller-Dieker Syndrome, a rare neurodegenerative disease in which one chromosomal copy of the mettl16 gene is deleted. Thus, METTL16 and the MALAT1 triple helix represent a model system with high potential to transform the field of triple- stranded RNA biology, opening unforeseen areas of biomedical investigation.